Submitted to: Molecular Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/16/2016
Publication Date: 10/18/2016
Citation: Yasmeen, A., Kiani, S., Butt, A., Rao, A.Q., Akram, F., Ahmad, A., Nasir, I.A., Husnain, T., Mansoor, S., Amin, I., Zubair, M., Tahir, M.N., Akhtar, S., Scheffler, J.A., Scheffler, B.E. 2016. Amplicon based RNA interference targeting V2 gene of cotton leaf curl Kokhran virus-Burewala strain can provide resistance in transgenic cotton plants. Molecular Biotechnology. doi:10.1007/s12033-016-9980-8.

Interpretive Summary: Cotton leaf curl virus is a devastating disease in Pakistan and is spreading to other countries. Millions of bales of cotton are lost each year due to this virus and there are currently no resistant varieties available. It may be possible to control the effects of the virus using new biotechnology methods, where a modified form of a gene is inserted into a cotton plant as a defense mechanism against the virus. When the virus invades the plant, it works by deactivating a viral gene (V2) that allows the virus to move within the plant and stops the virus from spreading. These new technological approaches will complement traditional methods and give the plant breeder more weapons to use in mitigating the effects of cotton leaf curl virus disease.

Technical Abstract: An RNAi based gene construct designated “C2” was used to target the V2 region of the cotton leaf curl virus (CLCuV) genome which is responsible for virus movement. The construct was transformed into two elite cotton varieties MNH-786 and VH-289. A shoot apex method of plant transformation using Agrobacterium (strain LBA4404) mediated transfer was performed. Transformation efficiency was 3.75% and 2.88 % for MNH-786 and VH-289 respectively. Eighty six plants were obtained after screening on MS medium containing 50 mg/µl kanamycin. Confirmation of successful transformation was done through PCR in the To, T1 and T2 generations using gene specific primers. The transgenic plants were evaluated for CLCuV disease in the T1 generation. Knockdown ability of the RNAi construct was confirmed through real time CLCuV titer PCR. The copy number and transgene location were observed by using Florescent in Situ Hybridization and karyotyping in T2 generation. The results of this study confirmed the integration of the RNAi gene construct in T0, T1 and T2 generations. Transformants with a single copy were identified for both varieties. A comparison of virus disease index score and the virus titer calculation from absolute quantification through real time PCR showed that the CLCuV tolerant transgenic cotton had minimal symptoms and low virus titer. These results demonstrate a new strategy to control CLCuV in cotton.